The hepatitis C virus (hereinafter, referred to as HCV) is a virus belonging to the flavivirus family the genome of which is a single-stranded (+) sense RNA and is known to cause hepatitis C. Based on recent studies, it has been revealed that HCV is classified into many types depending on genotype or serotype. According to phylogenetic analysis by Simmonds et al. using nucleotide sequences of HCV strains, HCV genotypes are classified into six types, and each type is further classified into several subtypes (Simmonds et al., Hepatology, (1994) Vol. 10, pp. 1321-1324). The full-length genomic nucleotide sequences of a plurality of HCV genotypes have also been determined to date (Choo et al., Science, (1989) Vol. 244, pp. 359-362, Kato et al., J. Med. Virol., (1992) Vol. 64, pp. 334-339, Okamoto et al., J. Gen. Virol., (1992) Vol. 73, pp. 673-679, Yoshioka et al., Hepatology, (1992) Vol. 16, pp. 293-299 and Mori et al., Biochem. Biophys. Res. Commun., (1992) Vol. 183, pp. 334-342).
Until recently, infection of cultured cells with HCV and replication of HCV genomes in cultured cells have been impossible. Accordingly, studies on mechanisms of HCV replication and infection have required in vivo experiments using chimpanzees as experimental animals. However, subgenomic replicon RNAs have been produced from the Con1 strain, the HCV-N strain, and the HCV-O strain belonging to the HCV genotype 1b, and the H77c strain belonging to the HCV genotype 1a, and this enabled studies on the HCV replication mechanism via in vitro experiments using cultured cells (JP 2001-17187 A, Lohmann et al., Science, (1999) Vol. 285, pp. 110-113, Blight et al., Science, (2000) Vol. 290, pp. 1972-1974, Friebe et al., J. Virol., (2001) Vol. 75, pp. 12047-12057 and Ikeda et al., J. Virol., (2002) Vol. 76, pp. 2997-3006). Herein, the subgenomic replicon RNA of HCV means an RNA which comprises a portion of HCV genome necessary for replication, and can autonomously replicate an RNA derived from the HCV genome when introduced into cultured cells, but does not have an ability to produce infectious HCV particles.
In addition, subgenomic replicon RNAs, and full-genomic replicon RNAs producing infectious HCV particles when introduced into Huh7 cells in vitro have been produced from the JFH-1 strain belonging to the HCV genotype 2a, and this enabled studies on the HCV infection mechanism via in vitro experiments using cultured cells (Kato et al., Gastroenterology, (2003) Vol. 125, pp. 1808-1817 and Wakita et al., Nature Medicine, (2005) Vol. 11, pp. 791-796). Herein, the full-genomic replicon RNA of HCV means an RNA which comprises a full-length HCV genome, i.e., a 5′ untranslated region, structural genes, non-structural genes, and a 3′ untranslated region, and can autonomously replicate an RNA derived from the HCV genome when introduced into cultured cells.
The J6CF strain is a HCV of genotype 2a, as is the JFH-1 strain. Homology between the J6CF strain and the JFH-1 strain is as high as about 90% at nucleic acid level and 91% at amino acid level. However, the J6CF strain lacks replication ability of its subgenomic replicon RNA, and it is not capable of virus particle production in a cell culture system using Huh7 cells.
Recently, substitution of an NS3 protein coding region, an NS5B protein coding region, and a 3′ untranslated region of the J6CF genome with the sequences of the same regions of the JFH-1 strain was found to lead to autonomous replication in Huh7 cells (Murayama et al., J. Virol., (2007) Vol. 81, pp. 8030-8040). In addition, the subgenomic replicon RNA and full-genomic replicon RNA prepared by substituting an NS3 protein coding region and a 3′ untranslated region of the J6CF genome with the sequences of the same regions of the JFH-1 strain and introducing three adaptive mutations in the NS5B protein coding region have been demonstrated to have autonomous replication ability and virus particle-producing ability (Murayama et al., PLoS Pathogens., (2010) Vol. 6, e1000885).
An experimental system that can efficiently amplify viruses is indispensable for studies on HCV and research and development of anti-HCV drugs. A system for amplifying HCV in cultured cells and a system for evaluating the propagation of HCV in cultured cells enable efficient screening of anti-HCV drugs. In addition, based on the systems, mechanisms of virus propagation will be understood, and new targets of anti-HCV drugs will be also found, and development of innovative anti-HCV drugs against such targets can be expected. Production of replicon RNA capable of autonomous replication in cultured cells or virus particle production is also desired for the J6CF strain.
Up to the present, however, it is believed that a full-genomic replicon RNA having infectious HCV particle-producing ability is required to comprise a sequence derived from the JFH-1 strain as a non-structural gene that is indispensable for virus replication. J6CF strain-derived full-genomic replicon RNAs autonomously replicating in cultured cells and producing HCV particles have been obtained, but they are limited to those composed of chimeric nucleic acids resulting from substitution of a considerable part of the non-structural genes in the J6CF genome with a sequence of the same region derived from the JFH-1 strain. Full-genomic replicon RNA having the J6CF genome as a backbone and having infectious HCV particle-producing ability is necessary for research of the J6CF strain, but has not yet been obtained.
As described above, subgenomic replicon RNA or full-genomic replicon RNA derived from HCV of genotype 2a corresponding to the replication mechanisms or replication efficiency of HCV strains other than the JFH-1 strain has not yet been obtained. Accordingly, it has been difficult to identify factors necessary for HCV replication serving as targets of new anti-HCV drugs or to screen for anti-HCV drugs capable of exerting drug efficacy independent of the replication mechanisms or replication efficiency. In addition, the types of artificially producible HCV particles serving as a raw material for HCV vaccine have been limited.
It could therefore be helpful to provide a replicon RNA having the genome of the hepatitis C virus J6CF strain as a backbone and having autonomous replication ability.